All publications cited herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art or relevant to the presently disclosure, or that any publication specifically or implicitly referenced is prior art.
Complete blood count (CBC) is a frequently used diagnostic test in clinics. It measures a blood sample for parameters such as leukocyte count, erythrocyte count, platelet count, hemoglobin concentration and hematocrit. It may also measure additional leukocyte parameters such as leukocyte differential (e.g., lymphocyte, monocyte, neutrophil, eosinophil, and basophil), reticulocyte count, nucleated erythrocyte count, erythrocyte indices (e.g., hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and red cell distribution width (RDW)), and platelet indices (e.g., mean platelet volume (MPV), plateletcrit (PCT), platelet distribution width (PDW), and platelet large cell ratio (PLCR)).
Testing of CBC is usually performed on automated hematology analyzers. These analyzers have a fluidic system that processes blood samples with different reagents and delivers them for sensing signal measurements. In conventional hematology analyzers, this fluidic system is built into the instrument for continuous use. After measuring one sample, the system needs to be flushed with a cleaning reagent to remove any residual sample. In point-of-care applications, an alternative format of the fluidic system is a disposable cartridge. The cartridge has on-board fluidics and is inserted into an instrument before testing a sample, and replaced after measurement. Each sample can be measured in a new cartridge, and there is no residual sample from the previous measurement.
U.S. Pat. Nos. 7,771,658 and 8,573,033 discuss methods of using a cartridge having on-board fluidics to perform the CBC test. In these methods, measurements of electrical impedance are used to detect the cells in the blood such as leukocytes, erythrocytes, and platelets. In comparison to this electrical impedance method, optical measurements are often preferred for CBC testing. For one reason, optical measurements achieve higher accuracy of CBC testing. Electrical impedance method can distinguish leukocyte cells into three subtypes including lymphocytes, monocytes and granulocytes, whereas optical method can distinguish leukocyte cells into five subtypes including lymphocytes, monocytes, and granulocytes, and can further distinguish granulocytes into neutrophil, eosinophil and basophil cells.
U.S. Pat. Nos. 8,741,233 and 8,741,234 discuss methods of using a cartridge having on-board fluidics to perform the CBC test with optical measurements. In these methods, a cytometer flow cell with a sheath flow design was used for the optical measurements. However, the sheath flow design is complicated and requires accurate control of flow rates to work properly. Furthermore, it delivers the sheath flow buffer together with the sample in the fluid channel, which makes the sample inaccessible to any direct measurement of the sample volume.
Flow cytometry is a method for accurately detecting and characterizing cells in biological samples. It is used in CBC testing such as counting and characterizing of leukocyte cells, erythrocyte cells and platelet cells. In conventional flow cytometers, the design of the flow cell also uses the sheath flow design. The flow cell usually uses a fluid channel with a size of several hundred of micrometers in diameter, which is significantly larger than the size of the blood cells (e.g., leukocyte cells 6-15 μm in diameter, erythrocyte cells 6-8 μm in diameter, and platelet cells 1-2 μm in diameter). To confine the sample into a narrower stream (e.g. 20-30 μm in diameter), which is important for accurate measurement of the cells, the flow cell uses the sheath flow, also known as hydrodynamic focusing. By passing an additional sheath flow together with the sample stream through the flow cell, the sheath flow confines the sample stream in the center into a narrowed stream. By adjusting the ratio of the flow rates of the sheath flow and the sample stream, the sample stream can be confined into any desired diameter for measurement. Complex fluidic structures are required to introduce the sheath flow and maintain the consistency of the flow rates. It is also difficult to control the volume of the sample being measured, and thus hard for this design to achieve measurement accuracy of the absolute count, which is the number of target particles per sample volume.
Shi et al. (Four-part leukocyte differential count based on sheathless microflow cytometer and fluorescent dye assay, Lab Chip. 2013 Apr. 7; 13(7):1257-65) discusses a method of using a cytometer flow cell with a sheathless design for the leukocyte differential information. In the sheathless design, the flow cell uses a fluid channel with small diameter and no sheath flow is used. This work teaches a method of measuring the percentages of each leukocyte subtypes (e.g. lymphocyte, monocyte, neutrophil, eosinophil and basophil) in the sample. But it does not teach how to directly measure the absolute count of the leukocyte, which is the number of target leukocyte cells per volume. Instead, it uses an indirect method: pipetting a fixed amount of blood sample to mix with reagents, and then measuring the full amount of this mixture for leukocytes. This method does not work if not the full amount of the mixture is measured. Additional, it does not teach how to measure other CBC parameter (e.g., erythrocyte count, platelet count, hemoglobin concentration, hematocrit, reticulocyte count, nucleated erythrocyte count, erythrocyte indices, and platelet indices).